Recombinant P. heparinus Heparinase III Protein, CF

Heparinase III digestion of Heparan Sulfate (200 μg) is assessed in a 5-minute kinetic assay atroom temperature by monitoring absorbance at 232 nm. R&D Systems Recombinant P.heparinum Heparinase III (Catalog # 6145-GH) (orange) exhibits much greater activity than the Bacteroides Heparinase III from the competition (green).

• Reactivity: Ph
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant P. heparinus Heparinase III Protein, CF Summary

• Details of Functionality: Measured by its ability to liberate oligosaccharides from heparin. The specific activity is >3,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived p. heparinus Heparinase III protein
  Gln25-Pro659, with an N-terminal Met and 6-His tag
• Accession #: AAB18278
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Phep_3797
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 74 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 66 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 2 mM CaCl₂, pH 7.5
  • Recombinant P. heparinus Heparinase III (rP. heparinus Heparinase III) (Catalog # 6145-GH)
  • Substrate: Heparin (Tocris, Catalog # 2812), 20 mg/mL stock in deionized water
  • 96 well clear UV-transparent microplate (Corning, Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rP. heparinus Heparinase III to 0.5 ng/µL in Assay Buffer.
  2. Dilute Substrate to 1.5 mg/mL in Assay Buffer.
  3. Load into a plate 100 µL of the diluted rP. heparinus Heparinase III, and start the reaction by adding 200 µL of 1.5 mg/mL Substrate. Include a Substrate Blank containing 100 µL of Assay Buffer and 200 µL of 1.5 mg/mL Substrate.
  4. Read in kinetic mode for 5 minutes at an absorbance of 232 nm.
  5. Calculate specific activity:

  Specific Activity (pmoles/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 3800 M^-1cm^-1
  ***Using the path correction 0.92 cm
  Note: the output of many spectrophotometers is in mOD. Per Well:
  • rP. heparinus Heparinase III: 0.05 µg
  • Substrate: 1.0 mg/mL

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant P. heparinus Heparinase III Protein, CF

• Heparinase III

Background

Heparan sulfate is a sulfated glycosaminoglycan with the repeating disaccharide units of -4HexA1,4GlcNAc beta 1-. It is usually attached to the protein cores of proteoglycans found on the cell membrane and in the extracellular matrix where it binds to a variety of protein ligands and regulates a wide range of biological activities (1, 2). Heparan sulfate has a domain structure where sulfated regions are interspaced with less or non-sulfated regions (3, 4). Heparin shares the backbone structure with heparan sulfate but contains no non‑sulfated regions. Heparinases are a family of lyases that release unsaturated oligosaccharides from heparin and heparan sulfate upon digestion (5). Heparinase I recognizes highly sulfated regions and is more specific for heparin. Heparinase II digests both heparin and heparan sulfate. Heparinase III prefers less-sulfated regions and is more active on heparan sulfate (6, 7).

1. MacArthur, J. M. et al. (2007) J. Clin. Invest. 117:153.
2. Esko, J. D. and Selleck, S. B. (2002) Annu. Rev. Biochem. 71:435.
3. Maccarana, M. et al. (1996) J. Biol. Chem. 271:17804.
4. Linker, A. and Hovingh, P. (1975) Biochim. Biophys. Acta. 385:324.
5. Linker, A. and Hovingh, P. (1965) J. Biol. Chem. 240:3724.
6. Su, H. et al. (1996) Appl. Environ. Microbiol. 62:2723.
7. Hovingh, P. and Linker, A. (1970) J. Biol. Chem. 245:6170.

Publications for Heparinase III (6145-GH)(11)

Publications using 6145-GH

• Chen, SJ;Lin, CM;Liu, CH;Chen, YH;Hsieh, YL;Lin, YC;Chu, YH;Ku, CY;Liao, WL;Liao, WC; Accelerated peripheral nerve repair using surface-modified biomaterials for targeted capture of glial-derived growth factors post-neurorrhaphy PloS one 2025-04-11 [PMID: 40215229] (Bioassay, Rat)
• Volpi, N;Galeotti, F;Gatto, F; High-throughput glycosaminoglycan extraction and UHPLC-MS/MS quantification in human biofluids Nature protocols 2024-11-14 [PMID: 39543382] (Bioassay, Human)
• Beddows, CA;Shi, F;Horton, AL;Dalal, S;Zhang, P;Ling, CC;Yong, VW;Loh, K;Cho, E;Karagiannis, C;Rose, AJ;Montgomery, MK;Gregorevic, P;Watt, MJ;Packer, NH;Parker, BL;Brown, RM;Moh, ESX;Dodd, GT; Pathogenic hypothalamic extracellular matrix promotes metabolic disease Nature 2024-09-18 [PMID: 39294371] (Bioassay, Mouse)
• Li, W;Plante, JA;Lin, C;Basu, H;Plung, JS;Fan, X;Boeckers, JM;Oros, J;Buck, TK;Anekal, PV;Hanson, WA;Varnum, H;Wells, A;Mann, CJ;Tjang, LV;Yang, P;Reyna, RA;Mitchell, BM;Shinde, DP;Walker, JL;Choi, SY;Brusic, V;Llopis, PM;Weaver, SC;Umemori, H;Chiu, IM;Plante, KS;Abraham, J; Shifts in receptors during submergence of an encephalitic arbovirus Nature 2024-07-24 [PMID: 39048821] (Bioassay, Human)
• YH Chu, WC Liao, YJ Ho, CH Huang, TJ Tseng, CH Liu Targeting Chondroitin Sulfate Reduces Invasiveness of Glioma Cells by Suppressing CD44 and Integrin beta1 Expression Cells, 2021-12-20;10(12):. 2021-12-20 [PMID: 34944101] (Bioassay, Human)
• M Puray-Chav, KM LaPak, TP Schrank, JL Elliott, DP Bhatt, MJ Agajanian, R Jasuja, DQ Lawson, K Davis, PW Rothlauf, Z Liu, H Jo, N Lee, K Tenneti, JE Eschbach, C Shema Mugi, EM Cousins, EW Cloer, HR Vuong, LA VanBlargan, AL Bailey, P Gilchuk, JE Crowe, MS Diamond, DN Hayes, SPJ Whelan, A Horani, SL Brody, D Goldfarb, MB Major, SB Kutluay Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell Cell Reports, 2021-06-23;0(0):109364. 2021-06-23 [PMID: 34214467] (Bioassay, Human)
• BL Benson, L Li, JT Myers, RD Dorand, UA Gurkan, AY Huang, RM Ransohoff Biomimetic post-capillary venule expansions for leukocyte adhesion studies Sci Rep, 2018-06-19;8(1):9328. 2018-06-19 [PMID: 29921896]
• Wu Z, Person A, Anderson M, Burroughs B, Tatge T, Khatri K, Zou Y, Wang L, Geders T, Zaia J, Sackstein R Imaging specific cellular glycan structures using glycosyltransferases via click chemistry. Glycobiology, 2018-02-01;0(0):. 2018-02-01 [PMID: 29186441] (Click Chemistry, Human, Mouse)
• S Schaller, D Buttigieg, A Alory, A Jacquier, M Barad, M Merchant, D Gentien, P de la Gran, G Haase Novel combinatorial screening identifies neurotrophic factors for selective classes of motor neurons Proc. Natl. Acad. Sci. U.S.A, 2017-03-07;114(12):E2486-E2493. 2017-03-07 [PMID: 28270618] (Bioassay, Mouse)
• Orme , Rowan P, Bhangal , Manminde, Fricker , Rosemary Calcitriol imparts neuroprotection in vitro to midbrain dopaminergic neurons by upregulating GDNF expression. PLoS ONE, 2013-04-23;8(4):e62040. 2013-04-23 [PMID: 23626767] (Bioassay, Rat)
• Wu ZL, Prather B, Ethen CM Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases. Glycobiology, 2010-12-17;21(5):625-33. 2010-12-17 [PMID: 21169395] (Enzyme Assay, Bovine, Porcine)

Bioinformatics

• Gene Symbol: Phep_3797
• Uniprot:
  • AAB18278()